Primary ciliary dyskinesia: evaluation using cilia beat frequency assessment via spectral analysis of digital microscopy images

Abstract

Ciliary beat frequency (CBF) measurements provide valuable information for diagnosing of primary ciliary dyskinesia (PCD). We developed a system for measuring CBF, used it in association with electron microscopy to diagnose PCD, and then analyzed characteristics of PCD patients. The CBF measurement system was based on power spectra measured through digital imaging. Twenty-four patients suspected of having PCD (age 1-19 yr) were selected from a group of 75 children and adolescents with pneumopathies of unknown causes. Ten healthy, nonsmoking volunteers (age ≥ 17 yr) served as a control group. Nasal brush samples were collected, and CBF and electron microscopy were performed. PCD was diagnosed in 12 patients: 5 had radial spoke defects, 3 showed absent central microtubule pairs with transposition, 2 had outer dynein arm defects, 1 had a shortened outer dynein arm, and 1 had a normal ultrastructure. Previous studies have reported that the most common cilia defects are in the dynein arm. As expected, the mean CBF was higher in the control group (P < 0.001) and patients with normal ultrastructure (P < 0.002), than in those diagnosed with cilia ultrastructural defects (i.e., PCD patients). An obstructive ventilatory pattern was observed in 70% of the PCD patients who underwent pulmonary function tests. All PCD patients presented bronchial wall thickening on chest computed tomography scans. The protocol and diagnostic techniques employed allowed us to diagnose PCD in 16% of patients in this study.

Fig. 1.

System for screening for ciliary beat frequency (CBF). Bottom left: power spectral density is a graph showing the power density as a function of the spectral component of the beat registered within the line of interest (LOI) with the predominant CBF accepted and registered. Bottom right: instantaneous luminance profiles along the LOI. Each profile is a graph of the temporal variation of luminance in a pixel contained within the LOI. Top: LOI. A line drawn on the sample image by the user (with the computer mouse) is shown, indicating the image pixels where the beat will be measured.

Fig. 2.

Cilia electron microscopy. Patients' cilia defects, which are marked with arrows, are as follows A: absence of dynein arms (magnification ×65,000, bar 200 nm); B: shortened outer dynein arm (magnification ×65,000, bar 200 nm); C: transposition defects (magnification ×65,000, bar 200 nm); and D: radial spoke with inner dynein arms defects (magnification ×30,000, bar 500 nm).

Fig. 3.

Cilia electron microscopy. Normal ultrastructure is shown (magnification ×39,000, bar 500 nm).